Key factors with ex-vitro acclimatization of tissue culture plants

Tissue culture is a primary propagation engine for new infusions of disease-free germplasm. Though commonplace for many species, a frequent challenge for tissue culture plants remains achieving high post-acclimatization (or hardening) survival and vigorous growth. While each tissue culture method used (micropropagation, organogenesis, embryogenesis or embryo rescue) has its quirks, the need is the same once removed from jar or tub and transplanted: Keep them alive and get the root and shoot growing rapidly.

The plant’s in vitro existence in the lab is physiologically a fat and lazy one if protocols are reasonably optimized. A tissue culture vessel is a closed, protective cocoon. It creates a near-saturated humidified environment (>95% RH), without air movement, and where light levels are generally moderate and controlled. Lastly, nutrients and water are freely provided for plant uptake via a semi-solid medium.

The ex vitro hardening process requires plants to adjust to much wider and more rapid swings in environmental conditions. This must be carefully managed by greenhouse and nursery growers in a stepwise manner. While vigorous species can manage this process with seeming ease, other species and sometimes specific cultivars can be much more challenged and require slower and/or longer periods of adjustment.

Plant quality

The natural tendency when problems arise is to focus on the acclimatization environment. Is it too dry, too wet, too bright, too warm or too cold? Most often the plant quality coming from the lab largely determines the outcome well before the plants are ever exposed to the external environment.

Quality at the lab

Development of cultural methods for a new cultivar or species from tissue culture often poses the eternal “chicken or egg” question. When early greenhouse outcomes are poor, “is it the greenhouse or the lab’s fault”?

The best method of resolution from the seemingly circular discussion is for the lab and greenhouse to collaborate. A useful first question can be, “What is the range in morphology that is currently coming to the greenhouse/acclimatization space?” If it is highly variable or not tracked, this suggests an opportunity for all involved to learn and to test. This can be done by planting plants with similar morphology and observing the result under current acclimatization conditions. If a morphotype is failing at a high frequency, and for obvious reasons (lack of general size, lack of foliage, lack of roots, hyperhydricity, contamination), then lab protocols need to improve and/or transplant specifications need to be made more stringent. Often the short-term answer (albeit painful) is to not ship or transplant low-quality material. While this is obvious for plant survival, it can also substantively impact final deliverable yield due to insufficient growth. This downstream loss can be very significant and costly. Understanding the full downstream cost implications of poor tissue culture stock often justify significant investments in process and infrastructure in the lab and acclimatization stages.

“Green plant with roots” is not a specification unless most everything transplanted survives and grows well.

Transplant selection at the greenhouse

“Green plant with roots” is not a specification unless most everything transplanted survives and grows well. In most cases, additional objective criteria are required: root length, stem length, leaf number, longest leaf length, etc. Note that there are at least two aspects of “the shoot”: the stem and the foliage. Length and caliper of the stem is critical during transplant, and is the conduit for water, sugar and nutrition transport. The foliage likewise can be broken down into the number of leaves, leaf size and net foliar surface area. The sum of plant foliar surface area largely determines the growth rate potential in well-rooted situations. Leaf and shoot characteristics tend to be broadly correlated, but not always, and particularly coming from artificial environments.

While the presence of a root is generally positive, there are some important caveats. Allowing roots to extend much past soil matrix depth can result in decline on the root-to-shoot ratio that can cause physiological stress if root trimming is severe.

Secondly, longer roots that are more vascularized (brittle) at their base are more prone to damage and challenging to transplant. For these reasons, planning for expected rooting duration in the lab schedule is very important, but should also not be set in stone. The lab should always visually verify a batch has attained transplant specifications before it is transferred to acclimatization. Finally, timely inspection and real-time feedback by greenhouse staff provides a final opportunity to catch issues for correction in the next production batch.

The ex vitro hardening process must be carefully managed by growers in a stepwise manner.

Despite best efforts, sometimes long roots just cannot be avoided. Root growth in some species or cultivars are just strongly favored over shoots in vitro. In this case other factors such as transplant media volume, soil media components and transplant method may need to be altered. This will be further discussed in the next installment of the series.

A plant is ultimately the sum of its parts, and because of this transplant decisions are made on plants, not individual plant parts. Use of large photo examples is a critical quality control tool for aiding transplanter decision-making. Photographs should have a sense of scale and display “ideal,” “minimally acceptable” and “clearly unacceptable” plants. Several examples of each category should be present. During routine discussion with staff, these pictures can be a very useful tool to help guide discussions and provide a common frame of reference and institutional memory of standards.

Frequent and well-communicated training and quality control feedback along the transplant line cannot be overemphasized. While it is obvious to provide feedback to new workers, experienced staff and management also benefit. Drift in acceptable quality can occur very slowly as lots from the lab can evolve over weeks and months without anyone taking notice until plant outcomes begin to mysteriously change. Discussion about quality supports both workers and management balance the competing goals of achieving high transplant rate versus maintaining transplant quality. In the final analysis, the combination of clear specifications, training and feedback will greatly improve the odds of greenhouse benches being filled with uniform, high-vigor trays that come from tissue culture origin.